U.S. patent application number 14/004235 was filed with the patent office on 2014-02-20 for method for the production of recombinant human factor viii.
This patent application is currently assigned to UNIVERSIDADE DE S O PAULO - USP. The applicant listed for this patent is Christian Colin, Marcos A. A. Demasi, Mari Cleide Sogayar. Invention is credited to Christian Colin, Marcos A. A. Demasi, Mari Cleide Sogayar.
Application Number | 20140051832 14/004235 |
Document ID | / |
Family ID | 46829968 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140051832 |
Kind Code |
A1 |
Demasi; Marcos A. A. ; et
al. |
February 20, 2014 |
METHOD FOR THE PRODUCTION OF RECOMBINANT HUMAN FACTOR VIII
Abstract
The object of the present invention is to provide methods for
the production of recombinant human Factor VIII, employing specific
endoproteases, thus assuring full proteolytic processing of said
factor even during its biosynthesis, consequently avoiding
additional purification steps. Other objects of the present
invention are the recombinant human Factor VIII as obtained by said
methods, pharmaceutical compositions, related uses and therapeutic
methods.
Inventors: |
Demasi; Marcos A. A.; (Sao
Paulo, BR) ; Sogayar; Mari Cleide; (Sao Paulo,
BR) ; Colin; Christian; (Sao Paulo, BR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Demasi; Marcos A. A.
Sogayar; Mari Cleide
Colin; Christian |
Sao Paulo
Sao Paulo
Sao Paulo |
|
BR
BR
BR |
|
|
Assignee: |
UNIVERSIDADE DE S O PAULO -
USP
Sao Paulo
BR
|
Family ID: |
46829968 |
Appl. No.: |
14/004235 |
Filed: |
March 11, 2011 |
PCT Filed: |
March 11, 2011 |
PCT NO: |
PCT/BR11/00069 |
371 Date: |
November 6, 2013 |
Current U.S.
Class: |
530/383 ;
435/69.6 |
Current CPC
Class: |
C07K 14/755 20130101;
C12Y 304/21826 20130101; C12N 9/6454 20130101; C12P 21/06
20130101 |
Class at
Publication: |
530/383 ;
435/69.6 |
International
Class: |
C07K 14/755 20060101
C07K014/755 |
Claims
1. Method for the production of recombinant human factor VIII
wherein comprises the introduction of coding regions for
endoproteases SPC6 and/or PACE4 AI and/or PACESOL into the genome
of animal cells producing Factor VIII and/or the addition of
endoproteases SPC6 and/or PACE4 AI and/or PACESOL to the growing
medium containing recombinant Factor VIII in a method comprising
partial deletions of the portion of the coding region for factor
VII corresponding to domain B and the independent production of
heavy and light chains, in which paths with variable extensions,
corresponding to domain B, are connected to the coding region for
the light chain.
2. Method according to claim 1 wherein the coding regions for
endoproteases SPC6 and/or PACE4 AI and/or PACESOL is stably
inserted in the genome of growing animal cells, so that such cells
may start to produce endoproteases, to be used, jointly or
separately, in the proteolytic processing of artificial forms of
Factor VIII, by the addition of endoproteases to the growing medium
containing cells producing recombinant human Factor VIII.
3. Method according to claim 1 wherein the production of human
endoproteases SPC6, PACE4 AI and PACESOL is effected by means of
the translation of their coding region in vitro, by means of
chemical synthesis or purification from naturally producing
cells.
4. Method according to claim 1 to wherein the endoproteases SPC6,
PACE4 Al or PACESOL have amino acid analogues or non-natural amino
acids in their sequences, keeping their endoproteolytic
activities.
5. Recombinant Human Factor VIII wherein it is obtained according
to the method of claim 1.
6.-12. (canceled)
Description
FIELD OF THE INVENTION
[0001] The object of the present invention is to provide methods
for the production of recombinant human Factor VIII, employing
specific endoproteases, thus assuring full proteolytic processing
of said factor even during or after its biosynthesis, consequently
avoiding additional purification steps. Other objects of the
present invention are the recombinant human Factor VIII as obtained
by said methods, pharmaceutical compositions, related uses and
therapeutic methods.
BACKGROUND OF THE INVENTION
[0002] Hemophilia is a genetic dysfunction disabling the body to
control bleedings (hemorrhagic diathesis), due to the reduction,
inactivation or lack of one of the coagulation factors as required
to form a blood clot.
[0003] Excessive bleeding may be external, if the skin is damaged
by a cut or abrasion, or internal, on muscles, articulations or
organs. Hematomas, hemarthrosis (bleedings on articulations) and
intracranial bleedings frequently appear and, in the lack of an
effective treatment, may even cause death.
[0004] The dysfunction may be classified into three types: [0005]
Hemophilia A--characterized by the lack of the coagulation Factor
VIII (FVIII), or anti-hemophilic globulin. [0006] Hemophilia B--or
Christmas disease, characterized by the lack of the hemophilic
factor B or factor IX. [0007] Hemophilia C--determined by a
dominant autosomic gene not related to gender, characterized by the
lack of a factor called PTA or factor XI.
[0008] Hemophilia A, characterized by the lack, inactivation or
partial activation of the coagulation Factor VIII, mainly affects
male patients, being the most common kind against diagnosed
patients.
[0009] The reduction of Factor VIII is also related to von
Willebrand's disease, which is another hereditary dysfunction
characterized by abnormal slow speed of blood coagulation, causing
spontaneous and long nose and gum bleedings, affecting both men and
women. Von Willebrand's factor is responsible for the protection
and stabilization of Factor VIII and, in its absence, there is a
consequent reduction of Factor VIII.
[0010] Human Factor VIII, which is essential for blood coagulation,
is a non-enzymatic glycoprotein present in blood plasma, acting in
the intrinsic route of the blood coagulation cascade, as a
co-factor of serine-protease factor IX, in the step of proteolytic
activation of factor X.
[0011] Patients with diseases related to the reduction or lack of
Factor VIII, such as hemophilia A, must increase the levels of
Factor VIII in the blood by means of administration of exogenous
Factor VIII, thus avoiding hemorrhagic occurrences and expontaneous
bleeding.
[0012] This is provided by a "factor reposition therapy", usually
made intravenously. Such treatment started in the 1950s, with
direct plasma transfusion, and later with the use of Factor VIII
concentrates, both obtained from plasma from blood donators.
[0013] During the 1970s and 1980s, the lack of other routes of
treatment and the lack of technology to diagnose a few viruses,
such as the Acquired Immunodeficiency Syndrome (AIDS) virus, caused
a large scale contamination in the hemophilic population.
[0014] Initially, concentrates obtained from human Factor VIII also
included contaminants, but technical advancements, added to the
adoption of a range of steps concerning the method to obtain them,
made their use become safer. However, besides the theoretical risk
still existent towards the eventual appearance of a pathogen (e. g.
a still not characterized virus, resistant against the viral
inactivation and purification methods as used), there are still
problems concerning the reduced quantity of donators and the very
low concentrations of Factor VIII as present in the plasma.
[0015] These facts make the method to obtain highly purified Factor
VIII preparations from donators' plasma become very complex and
expensive, not only due to the need of large quantities of human
plasma, a rarer and rarer material, but also for the complexity in
terms of quality control of the final product to be given to
patients with dysfunctions related to the reduction, inactivation
or lack of coagulation Factor VIII.
[0016] Since the 1990s, an alternative form of production of human
Factor VIII has been adopted, involving the stable transfection of
the nucleotide sequence corresponding to the protein-coding region
of the human Factor VIII gene into the genome of animal cells in
culture. Therefore, said cells start stably producing human Factor
VIII in culture and to secrete Factor VIII in the cultivation
medium, from which it may be purified later on.
[0017] Human Factor VIII as produced in this way is known as
recombinant human Factor VIII. In comparison with Factor VIII
concentrates as obtained from plasma from human donators,
recombinant human Factor VIII is considered as a potentially
non-exhaustive and safer source.
[0018] To obtain cells in culture producing recombinant human
Factor VIII, both the full coding region of the Factor VIII gene or
portions of the coding region which are enough to obtain a
functional product have been used.
[0019] The second-generation recombinant product, as commercialized
by the Wyeth group under the commercial name Re-Facto, is produced
by using two portions of the full coding region of human Factor
VIII connected by an artificial connection sequence. On the other
hand, first-generation recombinant products, commercialized by the
Bayer group under the commercial names Kogenate, Recombinate or
Helixate, are produced by using the full coding region of the human
Factor VIII gene.
[0020] The production of recombinant human Factor VIII from its
full coding region presents a range of obstacles, taking the
expression in an industrial platform to unsatisfactory levels.
[0021] One of the alternatives used to overcome such problem has
been the use of portions of the coding region of Factor VIII which
are enough to obtain a functional product. The most explored
changes are the partial or full deletion of a portion of the coding
region for Factor VIII, coding the central part of the molecule,
known as domain B. Said changes allow for higher production of
Factor VIII, although also industrially unsatisfactory (Saenko, E.
L., Ananyeva, N. M., Shima, M., Hauser, C. A. & Pipe, S. W.
(2003). The future of recombinant coagulation factors. J Thromb
Haemost, 1, 922-30).
[0022] In the full deletion of the portion of the coding region for
Factor VIII corresponding to domain B, coding regions for the
remaining portions of the molecule, known as heavy and light
chains, are connected by using an artificial connection sequence,
as used in the commercial product Re-Facto as mentioned above. Said
process also presents low industrial yields (Lind P., Larsson K.,
Spira J., Sydow-Backman M., Almstedt A., Gray E. and Sandberg H.
(1995). Novel forms of B-domain-deleted recombinant Factor VIII
molecules construction and biochemical characterization. Eur. J.
Biochem. 232, 19-27).
[0023] A second alternative involves partial deletions of the
portion of the coding region for Factor VIII corresponding to
domain B and the separate production of heavy and light chains of
Factor VIII. In this configuration, excerpts of variable
extensions, corresponding to domain B, are connected to the coding
region for light chain (U.S. Pat. No. 5,693,499, by Chemo Sero
Therapeut Res Inst (JP)).
[0024] Both in the full deletion and in the partial deletion as
disclosed above, synthesized products must be proteolytically
processed. In the case of full deletion, proteolytic processing is
required for the physical separation between heavy and light chains
and the formation of active Factor VIII to occur. In the case of
partial deletion, however, the proteolytic processing of the light
chain fused to the excerpt of domain B must occur, so that the
light chain as produced is identical to the one as naturally found
in human plasma.
[0025] Proteolytic processing also occurs naturally, even when the
full coding portion is used, i. e. mammal cells as used to produce
Factor VIII have the ability to proteolytically process Factor
VIII, despite not knowing which enzyme is responsible for said
activity. However, this capacity is very limited, i. e. when said
cells produce higher quantities of Factor VIII, said proteolytic
processing is always partial.
[0026] No matter which is the used alternative, the main problem is
still the partial or incomplete proteolytic processing of the
products as formed. This causes the production of artificial forms
of Factor VIII (Factor VIII complex) which, if present in the final
pharmaceutical product, could present unexpected antigenic
properties to the patient to be treated.
[0027] Furthermore, said incomplete processing reduces the quantity
of the final product as obtained, considering that part of what is
being produced by the animal cells is being converted into a
subproduct, i. e. into artificial forms of Factor VIII which need
to be excluded in some part of the production method.
[0028] This not only results in less yielding of the final product,
but also in a longer and more expensive method.
[0029] Causes of such an incomplete proteolytic processing have not
been fully clarified. A few hypotheses would be: a) saturation of
protease(s) as naturally present in cells used to produce large
quantities of Factor VIII; or b) eventual differences in activity
and specificity between protease(s) as present in non-human cells
used to produce Factor VIII and human protease(s) naturally
performing said processing.
[0030] According to the hypothesis b) above, although cells used to
produce Factor VIII are able to proteolytically process said
factor, they do so only partially, since said cells do not have
protease(s) naturally performing said processing.
[0031] Therefore, considering the possibility of partial or
incomplete proteolytic processing, the process to produce
recombinant Factor VIII will be longer, since it always requires a
purification step for control of eventual forms derived from Factor
VIII.
[0032] The technology proposal for full proteolytic processing has
been little explored, since there is full specificity between
endoprotease and precursor, making it difficult and costly to
search which endoprotease is able to process a given precursor.
Furthermore, theoretical methods are not efficient to indicate
possibly effective endoprotease(s). This means that, despite a
given protein theoretically has a site recognized by endoproteases,
it is not possible to realize which one would be able to
effectively recognize said site.
[0033] The state of the art teaches the use of different
endoproteses in the proteolytic processing of different blood
coagulation factors, confirming the specificity between
endoprotease and precursor, but lacking to propose an effective
means to guarantee the full proteolytic processing of human
coagulation Factor VIII, as previously explained.
[0034] Patent U.S. Pat. No. 5,965,425 by Genetics Inst (US),
published on Oct. 12, 1999, teaches the use of specific
endoprotease in a method to increase the efficiency of proteolytic
processing of precursor polypeptides for factor VII, factor IX,
factor X, protein C, protein S, prothrombin and von Willebrand's
factor, in recombinant host cells, aiming to guarantee a correct
and efficient fold, thus providing increase in activity.
[0035] The international patent application WO 03/100053, by the
Philadelphia Children Hospital, published on Dec. 4, 2003, teaches
the use of an artificial site recognized by a specific
endoprotease. More specifically, variants as disclosed have full
deletion of the DNA sequence corresponding to the domain B and use
the sequences corresponding to heavy and light chains, or their
portions, interconnected by a coding DNA segment for an artificial
cleaving site of the specific endoprotease. Concerning the heavy
chain, the coding region of the natural heavy chain (amino acids
1-470) or their variants are used, causing small deletions of the
C-terminal end of heavy chain (amino acids 1-700, 1-710, 1-720 and
1-730). Concerning the light chain, variants of the coding region
for the light chain containing small deletions corresponding to the
N-terminal end of the light chain are used. Most of these variants
correspond to the amino acid sequence 1690-2232 and the other
variants are little smaller portions than this sequence (amino
acids 1700-2232, 1710-2232, 1720-2232 and 1730-2232).
[0036] The international patent application WO 2008/022151, by
Inspiration Biopharmaceuticals (US), published on Feb. 21, 2008,
discloses methods to produce factor IX by using the specific
endoprotease PACE and enzymes VKOR and VKGC as required for the
carboxylation of factor IX depending on vitamin K.
[0037] Therefore, there is still the need of enhancements to the
production process for recombinant human Factor VIII, so to
guarantee the full proteolytic processing, thus avoiding additional
purification steps.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A, 1B and 1C refer to configurations of the coding
region for Factor VIII as used for its production.
[0039] FIG. 2 schematically and simply shows a process to produce
separate heavy and light chains to obtain recombinant Factor VIII,
wherein (*) shows the Factor VIII complex containing the light
chain fused to the path of domain B due to incomplete proteolytic
processing.
[0040] FIG. 3 schematically shows a part of the process to produce
heavy and light chains to obtain Factor VIII, wherein B' highlights
the step guaranteeing full proteolytic processing in an embodiment
of the present invention.
[0041] FIG. 4 schematically shows part of the process to produce
heavy and light chains to obtain Factor VIII, wherein C' highlights
the step guaranteeing full proteolytic processing in an embodiment
of the present invention and (*) shows the Factor VIII complex
containing the light chain fused to the path of the domain B due to
incomplete proteolytic processing.
[0042] FIGS. 5 and 6 show a representative result corresponding to
the assays as made to evaluate the alternative for the simultaneous
production of Factor VIII and endoproteases, in an embodiment of
the present invention.
[0043] FIGS. 7 and 8 show a representative result corresponding to
the assays made to evaluate the alternative to produce proteases in
other cells and add them to the growing medium of cells producing
Factor VIII.
DISCLOSURE OF THE INVENTION The Applicant has verified that three
specific endoproteases are able to recognize natural sites existent
in the link between domains B and the light chain.
[0044] Therefore, it has been possible to develop enhanced
processes to produce recombinant human coagulation Factor VIII.
[0045] Therefore, the present invention refers to the use of
specific enzymes, notably endoprotease PCSK5--Protein Convertase
Subtilisin/Kexin type 5, also known as SPC6, and/or the isoform and
endoprotease PCSK6--Protein Convertase Subtilisin/Kexin type 6,
also known as PACE4--Paired basic Amino acid Cleaving Enzyme 4
and/or secreted mutant from the endoprotease PACE--Paired basic
Amino acid Cleaving Enzyme, known as PACESOL, so to promote a full
proteolytic processing of Factor VIII, still during the step of its
production by animal cells, thus avoiding additional steps of
purification and providing for enhanced methods to produce
recombinant human coagulation Factor VIII.
[0046] Endoproteases PACE, SPC6 and PACE4 belong to a family of
proteins acting in the processing of cell proteins which are
initially synthesized as latent precursors (inactive forms), but
need a portion of the molecule to be proteolytically removed to
cause the production of its mature (active) form. Until now, only a
few proteins which need SPC6 to be proteolytically processed are
known. These proteins are subunits alpha of some proteins known as
integrins. SPC6 seems to be also able to process proteins
pro-renin, pro-matrix metalloprotease type 1 and glycoprotein gp160
of the HIV virus. Concerning PACE4, proteins of the growth
transforming factor family, known as TGFp.beta.--Transforming
Growth Factor .beta., pro-albumin and Von Willebrand's factor are
examples of proteins which are proteolytically processed by said
endoprotease. At least eight alternative transcripts linked to the
human PACE4 gene are known, which code different isoforms
designated as isoforms a to h.
[0047] PACESOL is an artificial mutant form of the protein PACE
which does not have the residues 716-794. The
[0048] PACE protein is a member of the enzyme family able to Clive
after paired basic amino acid residues.
[0049] Patent number U.S. Pat. No. 5,965,425 as mentioned above
discloses the use of the endoprotease PACE and secondarily its
variants, like the secreted mutant PACESOL, to promote the process
of precursor proteins for some components of the coagulation
cascade, especifically factor IX, protein C, protein S,
prothrombin, factor X, factor VII and von Willebrand's factor,
aiming to obtain them with an appropriate fold in its biologically
active or mature forms. Coagulation Factor VIII has, in its
polypeptidic sequence, the sequence of residues of amino acid
Arg-Hys-Gln-Arg, which would theoretically be recognized by
endoprotease PACE. Therefore, in a first moment, the extension of
use of PACE for the promotion of proteolytic processing of Factor
VIII could be expected. However, endoprotease components of the
family of proteins of which PACE is a part present a few overlaying
examples for given substrates, such as in the case of von
Willebrand's factor, which is cleaved by both PACE and PACE4. In
other cases, there is some specificity e.g. for factor IX, which
apparently is only cleaved by PACE. However, in opposition to the
art, the Applicant concluded that the wild PACE enzyme does not
promote the cleavage of the junction between domain B and the light
chain, as could be expected by analogy, disregarding the use of
PACE and its variants in a first moment and confirming the
knowledge that it is not usual to anticipate by which
endoprotease(s) a given precursor may be processed.
[0050] Surprisingly, the Applicant verified that specific
endoproteases SPC6, isoform a of PACE4 (PACE 4 AI) and PACESOL are
able to promote the proteolytic process of recombinant human Factor
VIII. Therefore, from this knowledge, it was possible to enhance
methods to produce recombinant human Factor VIII, thus guaranteeing
full proteolytic processing and avoiding an additional step of
purification of Factor VIII complexes as eventually formed.
[0051] According to the present invention, "full proteolytic
processing" is the non-formation of a substantial quantity of
recombinant human coagulation
[0052] Factor VIII complexes containing the light chain fused to
the path of domain B, as shown by FIGS. 2 and 4, thus avoiding
additional purification steps.
[0053] By "substantial", we understand a quantity which is not
liable to cause damage to the patient, allowing eliminating an
additional purification step.
[0054] The method of the present invention involves partial
deletions of the portion of the coding region for Factor VIII
corresponding to domain B and the independent production of heavy
and light chains, as disclosed by the patent U.S. Pat. No.
5,693,499, incorporated herein as a reference. In this
configuration, excerpts of variable extensions, corresponding to
domain B, are connected to the coding region for the light
chain.
[0055] The coding sequence for the light chain as used in the
present invention corresponds to the natural non-processed amino
acid sequence of the light chain (amino acids 1648-2232) linked to
a path corresponding to a portion of domain B (amino acids
1563-1649). Said sequence has a natural site, adjacent to the amino
acid 1648, recognized by pro-convertase type endoprotease(s), i. e.
one or more members of the furin family.
[0056] The method of the present invention consists in (a)
introduction of coding regions for endoproteases SPC6 and/or PACE4
AI and/or PACESOL in the genome of animal cells producing Factor
VIII and/or (b) addition of endoproteases SPC6 and/or PACE4 AI
and/or PACESOL to the growing medium containing recombinant Factor
VIII.
[0057] In the method of the present invention, the portions of
coding regions corresponding to the heavy chain of human Factor
VIII and the light chain containing a small path corresponding to
domain B of the human Factor VIII are stably inserted in the genome
of growing animal cells, so that these cells start co-producing
heavy and light chains of the human Factor VIII and therefore large
quantities of the complex between heavy and light chains, with
coagulant activity (Factor VIII).
[0058] Besides the above elements, in a first embodiment of the
present invention, as shown by FIG. 3, coding regions for
endoproteases PCSK5 (SPC6) and/or the isoform a of PCSK6 (PACE4 AI)
and/or PACESOL are stably inserted in the genome of growing animal
cells producing the recombinant human Factor VIII, so that such
cells start to produce recombinant Factor VIII identically to what
is naturally found in human plasma and free from artificial forms
of Factor VIII (step B').
[0059] In a second embodiment of the present invention, as shown by
FIG. 4, coding regions for endoproteases SPC6 and/or PACE4 AI
and/or PACESOL are stably inserted in the genome of growing animal
cells, so that such cells may start to produce endoproteases PCSK5,
PACE4 AI or PACESOL, to be used, jointly or separately, in the
proteolytic processing of artificial forms of Factor VIII, by the
addition of endoproteases to the growing medium containing cells
producing recombinant human Factor VIII (step C').
[0060] According to the present invention, the production of
endoproteases SPC6, PACE4 AI or PACESOL to be used for the
promotion of the proteolytic processing of
[0061] Factor VIII can be made in a wide variety of growing cells
derived from animals, insects, fungi/yeasts and bacteriae as known
in the art.
[0062] Alternatively, the production of human endoproteases SPC6,
PACE4 AI and PACESOL may be effected by means of a cell-free
system, e. g. by translating its coding region in vitro, by means
of chemical synthesis of its purification from naturally producing
cells.
[0063] In another embodiment, endoproteases SPC6, PACE4 AI and
PACESOL may be produced so to have, in their sequences, non-natural
amino acids or amino acid analogues, as long as keeping their
endoproteolytic activities. In another embodiment of the present
invention, steps B' and C' may be additively performed.
[0064] As a result, the present invention provides an enhanced
method wherein there is the production of Factor VIII free from
artificial forms presenting unpredictable and more effective
antigenic potential, by the conversion of an eventually undesired
subproduct, which should be eliminated in some step of the
production method, into a Factor VIII which is identical to the one
as naturally found in human plasma. According to the present
invention, full coding regions of human endoproteases SPC6, PACE4
AI and PACESOL may be used or, alternatively, other configurations
of the coding regions for endoproteases SPC6, PACE4 AI and PACESOL
endoproteases, be them natural or artificial, may be used. In this
embodiment, coding regions for SPC6, PACE4 AI and PACESOL which are
different from the coding region concerning natural allelic
variations or variants from other species may be used, as well as
natural alternative forms of SPC6, PACE4 AI and PACESOL having the
same proteolytic activity, such as isoforms caused by an
alternative transcription of said genes and various
post-translational processes, such as proteolytic processing.
[0065] In this sense, coding regions for endoproteases SPC6, PACE4
AI and PACESOL from other animal species, be them mutant, natural
or artificial, may be used, as well as excerpts from the coding
region for these genes, with ability to generate truncated
polypeptides, fused or not to other polypeptides, but keeping the
same activity of original proteins.
[0066] According to the present invention, as the promoting region
for gene transcription, in case of production of light and heavy
chains of Factor VIII, the hybrid promoter partly derived from the
.beta.-actin gene of chickens and also from the transcription
enhancer derived from human cytomegalovirus is used. In case of the
production of endoproteases SPC6, PACE 4 AI and PACESOL, the
promoter derived from human cytomegalovirus is used.
[0067] Alternatively, with no limitation whatsoever, any strong
promoting region, natural or artificial, which is compatible with
the super production of the recombinant protein of interest, may be
used. Examples of other strong natural promoters which may be used
include initial and late promoting regions derived from the SV40
virus, the promoting region derived from the late region of
adenovirus and the Elongation Factor-1 (EF-1) gene promoter. An
example of an artificial promoting region includes SCP-1 (Super
Core Promoter-1). Furthermore, various transcription enhancers may
be linked to the promoting regions as disclosed above, such as the
transcription enhancing regions derived from murine polyoma and
SV40 viruses.
[0068] Particularly, animal cells of the present invention are from
the type CHO-DG44 of hamster ovary, which lack the enzyme
di-hydrofolate reductase (DHFR).
[0069] Furthermore, for the selection of cells having their
expression vector stably integrated in their genome, various
classes of selection markers may be used, being particularly
preferred selection markers coding proteins giving resistance
against some antibiotics, such as neomicin phosphotransferase and
hygromicin phosphotransferase and/or amplifiable selection markers
such as the sequences coding enzymes DHFR (dihydrofolate reductase)
and glutamine sinthetase.
[0070] For the production of endoproteases or light and heavy
chains of Factor VIII, the expression vectors do not necessarily
need to be stably integrated in the genome of growing cells.
[0071] In a particular embodiment, the production of endoproteases
may be reached by means of transitory insertion of the expression
vector into the nucleus of animal cells. In this case, the
production of endoproteases or heavy and light chains of Factor
VIII is also transitory.
[0072] In another embodiment, the insertion of elements into an
expression vector may be conducted, so to allow it, when in the
nucleus of animal cells, to be stably replicated and propagated not
being integrated to their genome.
[0073] Vectors of the present invention may contain various
configurations, e. g. one single expression vector containing
expression units from both the light chain and one of the used
endoproteases, as well as independent vectors for each element to
be expressed.
[0074] Without any limitation, according to the present invention,
the introduction of the expression vector for endoproteases into
the growing cells of interest may be made by employing any
appropriate method, being particularly preferred: direct
absorption, transfection mediated by calcium chloride, transfection
mediated by electroporation, lipofection and viral
transduction.
[0075] Still according to the present invention, endoproteases
SPC6, PACE4 AI and PACESOL may be produced in cells CHO-DG44 and
later used in the growing medium of cells super-producing Factor
VIII to promote proteolytic processing of the light chain or,
alternatively, cells producing endoproteases may be used in other
configurations to promote proteolytic processing of the light chain
of Factor VIII, e. g. in co-cultivation with cells super-producing
Factor VIII.
[0076] Therefore, with no limitation whatsoever, the present
invention includes the proteolytic processing of recombinant human
Factor VIII comprising the introduction of coding regions for
endoproteases SPC6 and/or PACE4 AI and/or PACESOL in the genome of
animal cells producing Factor VIII and/or the addition of
endoproteases SPC6 and/or PACE4 AI and/or PACESOL to the growing
medium containing recombinant Factor VIII.
[0077] In a particular embodiment, endoproteases SPC6, pACE4 AI or
PACESOL may be used for the proteolytic processing of other
recombinant Factor VIII configurations containing coding regions
for light and heavy chains of Factor VIII, interconnected through
an artificial connection region or interconnected with paths
corresponding to the domain B of varied extensions as disclosed by
Lind et al, 1995 (cited above).
[0078] The present invention also includes recombinant human Factor
VIII, which is obtained by the methods of the present invention, as
well as its use to prepare a pharmaceutical product for the
treatment of diseases related to the reduction or lack of Factor
VIII, like hemophilia A.
[0079] The pharmaceutical product of the present invention is
formulated with pharmaceutically acceptable excipients as known in
the art, for oral or injectable administration.
[0080] Pharmaceutically acceptable carriers to make the present
invention may include all those known in the art, with no
limitation whatsoever. A reference work for the formulation of said
pharmaceutical forms is the book Remington's Pharmaceutical
Sciences, from the U. S. publisher Mack Publishing.
[0081] Excipients are particularly selected from one or more among
histidine, Tris, BIS-Tris Propane, PIPES, MOPS, HEPES, MES, ACES,
sodium chloride, calcium chloride, calcium gluconate, calcium
glubionate, calcium gluceptate, glutathione, homocysteine, trolox,
lipoic acid, methionine, sodium thiosulphate, platinum, glycine,
BHT, polysorbate, Pluronic polyols, Brij, mannitol, alanine,
hydroxyethyl amide, sucrose, trehalose, raffinose, arginine,
albumin, etc.
[0082] Furthermore, an object of the present invention is to
provide methods to treat diseases related to the reduction or lack
of Factor VIII, particularly hemophilia A, consisting in the supply
to a patient in need of a therapeutically effective quantity of
recombinant human Factor VIII which is obtained following the
methods of the present invention, particularly in the form of a
pharmaceutical product.
[0083] The examples below serve to show aspects of the present
invention, not having, however, any limitative purpose.
EXAMPLES
Enzyme Selection
[0084] Six enzymes have been tested, PACE (or furin, abbreviated
FUR in the results as presented herein), PACESOL, a new isoform of
PACE4 still not characterized (abbreviated as PACE4 in the results
as presented herein) having the main domains responsible for PACE4
proteolytic activity, isoform a of PACE4 (abbreviated as PACE4 AI
in the results as presented herein), SPC6 and PCSK7, but only SPC6,
PACE4 AI and PACESOL show appropriate results, as shown by the
examples below.
Production Method
[0085] The method started from growing animal cells producing
recombinant human Factor VIII in large quantities. To obtain such
cells, coding regions corresponding to the heavy chain of Factor
VIII (amino acid residues 1 to 740, preceded by 19 amino acid
residues corresponding to the signal peptide of Factor VIII) and
the light chain containing a small path corresponding to domain B
of Factor VIII (residues 1,563 to 2,332, preceded by 19 amino acid
residues corresponding to the signal peptide of Factor VIII) have
been stably inserted into the genome of hamster ovary cells
CHO-DG44, which lack the enzyme di-hydrofolate reductase
(DHFR).
[0086] Coding regions as used need to be in a given configuration
to be functional in animal cells. Therefore, it is required that
coding regions are linked to regulatory elements causing the cell
biosynthetic machinery to effectively synthesize the proteic
product of interest. For the production of both the heavy chain and
the light chain, the following regulatory elements have been
used:
[0087] a) hybrid promoter region composed by the gene transcription
promoter of the .beta.-actin gene of chickens and the transcription
enhancer derived from human cytomegalovirus;
[0088] b) region of the internal link to the ribosome, known as
IRES (Internal Ribosome Entry Site), derived from the
encephalomiocardite virus of rabbits. The use of this region allows
to link the expression of the coding region of interest to the
expression of the selection marker of cells containing the elements
of interest integrated to its genome. In our case, the selecion
marker as used was the coding region of the enzyme DHFR of
mice;
[0089] c) A signaling region for the polyadenylation of the
transcript or mRNA (messenger RNA) generated from the transcription
of the coding region of interest. In our case, the signaling region
for polyadenylation derived from the .beta.-globin gene of rabbits
has been used.
[0090] The coding region of interest, linked to the regulatory
elements as disclosed above, constitutes an expression vector of
the coding region of interest. Expression vectors of light chain
and heavy chain have been co-introduced into CHO-DG44 cells by
means of a method known as lypofection.
[0091] The following step involved the selection, by means of
biochemical complementation, of CHO-DG44 cells stably expressing
the enzyme DHFR and consequently containing expression vectors of
interest as stably integrated to its genome.
[0092] This first cell population, containing expression vectors of
interest stably integrated to its genome, has been used to obtain
cells by producing Factor VIII in large quantities, by means of a
method known as gene co-amplification.
[0093] In that method, initially selected cells are submitted to
growing concentrations of an inhibitor for the enzyme DHFR,
methotrexate (MTX), thus selecting, at the end of each selection
cycle, cells producing growing quantities of the enzyme DHFR.
[0094] Since the production of the enzyme DHFR is linked to the
production of light and heavy chains of Factor VIII, the production
of said proteins also increases simultaneously.
[0095] Therefore, at the end of the method, CHO-DG44 cells have
been obtained, thus producing large quantities of the recombinant
human Factor VIII.
[0096] Cells producing recombinant Factor VIII as obtained present,
as an inconvenience, incomplete proteolytic processing of the light
chain.
[0097] To induce the proteolytic processing of the light chain, an
expression vector for human endoprotease SPC6 (PCSK5), an
expression vector for the isoform a of human endoprotease PACE4
(PCSK6) or an expression vector for the mutant as secreted from the
human endoprotease PACE, known as PACESOL, has been stably
introduced into the genome of CHO-DG44 cells super-producing Factor
VIII.
[0098] Expression vectors for SPC6, PACE4 AI and PACESOL as used
were lentiviral bicistronic expression vectors. Said vectors have
elements derived from the HIV (Human Immunodeficiency Virus) virus
causing their stable and very effective integration into the genome
of target cells. The promoting region of the gene transcription as
present in said vector is derived from the human
cytomegalovirus.
[0099] In that vector, the transcription of the coding region of
interest is linked to the transcription of the coding region of a
fluorescent protein known as EGFP (Enhanced Green Fluorescent
Protein). Said link is provided by the same element IRES as
disclosed above.
[0100] The fluorescent protein EGFP has been used as an indicator
of the stable integration of expression vectors as used in the
genome of cells super-producing Factor VIII.
[0101] The signaling region for polyadenylation as used was the
region known as LTR (Long Terminal Repeat) of the HIV virus.
CHO-DG44 cells super-producing Factor VIII thus modified
additionally produce recombinant human SPC6 endoprotease or
recombinant human PACE4 AI endoprotease or recombinant human
PACESOL endoprotease, and started to fully and proteolytically
process the light chain of Factor VIII fused to a small path of
domain B, making said modified cells to produce just the light
chain of Factor VIII identically to what is naturally found in the
human plasma, lacking antigene artificial forms.
[0102] In another test, the same expression vector for endoprotease
SPC6, the same expression vector for endoprotease PACE4 AI and the
same expression vector for endoprotease PACESOL have been stably
integrated into the genome of CHO-DG44 cells. Said cells started to
stably produce and secrete recombinant SPC6, PACE4 AI or PACESOL to
the growing medium. Recombinant endoproteases SPC6,
[0103] PACE4 AI and PACESOL thus produced have been added as
reagents to the growing medium of CHO-DG44 cells super-producing
Factor VIII, having also been able to promote, in vitro, the full
proteolytic processing of the light chain of Factor VIII.
[0104] Assays to Evaluate Proteolytic Processing
[0105] FIGS. 5 to 8 are representative of the assay made to
evaluate if the proteolytic processing of the light chain of Factor
VIII was occurring or not.
[0106] FIGS. 5 and 6 correspond to the assays made to evaluate the
alternative of simultaneous production of Factor VIII and
proteases.
[0107] FIGS. 7 and 8 correspond to the assays to evaluate the
alternative to produce proteases in other cells and add them to the
growing medium of cells producing Factor VIII. In said assays, PACE
(or furin) and PCSK7 enzymes have not been used, due to limitations
in their solubilities.
[0108] The assay as used is known as Western Blot and, in summary,
involves, in a first step, the fractioning of a mixture of
proteins, exploring the differences in molecular weight between
them and, in a second step, the detection of a given protein by
using specific antibodies against it.
[0109] In the assay as effected, the protein mixture was provenient
from the growing medium as conditioned by the cells producing
Factor VIII. Therefore, said growing medium contains, among other
components, synthesized proteins secreted by cells producing Factor
VIII, which is also secreted to the growing medium. The protein
which has been detected in the present assay was the light chain of
Factor VIII.
[0110] On FIGS. 5 to 8, each numbered vertical band represents an
analyzed mixture of proteins. The vertical band indicated as PM
corresponds to a mixture of proteins with known molecular masses
used as molecular weight standards.
[0111] To the left of the figures, the position of said molecular
weight markers is indicated (49 kDa and 180 kDa band). Bands 2 at
each figure correspond to the positive control for the detection of
the light chain of Factor VIII.
[0112] As a positive control, a commercial preparation of purified
human Factor VIII from donators' plasma has been used. In this
control, the detection of a proteic band of approximately 80 kDa,
corresponding to the fully processed light chain, is expected.
[0113] As one can verify in the results presented in the FIGS. 5
and 6, gutters 3 correspond to the samples of medium conditioned by
cells super-producing human Factor VIII, designated as H6A. In
those samples, a proteic band has been detected, at the same height
of the proteic band as detected in the positive control,
corresponding to a fully processed light chain.
[0114] A proteic band of about 100 kDa, corresponding to a
proteolytically non-processed light chain, has also been
detected.
[0115] Bands numbered as 5 to 9 on FIGS. 5 and 6 correspond to
samples of medium conditioned by genetically modified H6A cells so
to produce endoproteases PACE (bands 5, indicated as H6A-FUR-EGFP),
the secreted PACE mutant known as PACESOL (bands 6
H6A-PACESOL-EGFP), a new isoform of PACE 4 not yet characterized
(band 7 H6A-PACE4-EGFP of FIG. 5), isoform a of PACE 4 (band 8
H6A-PACE4 AI-EGFP of FIG. 6), SPC6 (bands 8 H6A-SPC6-EGFP) and
PCSK7 (bands 9 H6A-PCSK7-EGFP). Bands 4 of FIGS. 5 and 6 correspond
to the samples of medium as conditioned by genetically modified H6A
cells only with the expression vector of endoproteases
(H6A-EGFP).
[0116] As one can verify on FIGS. 5 and 6, the 100 kDa proteic band
corresponding to the non-processed light chain in medium
conditioned by H6A-PACESOL-EGFP (bands 6 of FIGS. 5 and 6),
H6A-PACE4 AI-EGFP (band 7 of FIGS. 6) and H6A-SPC6-EGFP (bands 8 of
FIGS. 6 and 6) cells has disappeared, indicating that the
proteolytic processing of the light chain is taking place in those
cells.
[0117] On FIG. 7, the gutter 3 corresponds to a sample of medium as
conditioned by H6A cells super-producing human VIII factor, wherein
the same profile as disclosed above has been detected for FIGS. 5
and 6.
[0118] Bands with numbers 6 and 7 correspond to samples of medium
conditioned by H6A cells growing with medium conditioned by
genetically modified parental cells (used to originate H6A cells)
CHO-DG44 to produce and secrete endoproteases PACESOL (band 6
H6A+CHO-DG44-PACESOL-EGFP) and a new and not yet characterized
isoform PACE 4 (band 7 H6A +CHO-DG44-PACE4-EGFP) to the growing
medium.
[0119] Band 4 corresponds to the medium conditioned by H6A cells
growing with the medium conditioned by cells CHO-DG44 (H6A
+CHO-DG44).
[0120] Band 5 corresponds to the medium conditioned by H6A cells
growing with the medium conditioned by genetically modified
CHO-DG44 cells only with the expression vector of endoproteases
(H6A+CHO-DG44-EGFP).
[0121] As one can observe on FIG. 6 that the 100 kDa proteic band
corresponding to non-processed light chain just in the medium
conditioned by H6A cells growing with the medium conditioned by
CHO-DG44-PACESOL-EGFP cells (band 6) has disappeared, indicating
that, upon the addition of PACESOL, in this case produced by
another cell line, to the growing medium of cells producing Factor
VIII, proteolytic processing of the light chain takes place.
[0122] On FIG. 8, the gutter 3 corresponds to a sample of medium as
conditioned by H6A cells growing with the medium as conditioned by
CHO-DG44 cells, wherein the same profile as disclosed above has
been detected for FIGS. 5, 6 and 7.
[0123] Bands with numbers 5 and 6 correspond to samples of medium
conditioned by H6A cells growing with medium conditioned by
genetically modified CHO-DG44 cells to produce and secrete a new
and yet not characterized isoform of PACE4 (band 5 H6A
+CHO-DG44-PACE4) and endoprotease SPC6 (band 6 H6A +CHO-DG44-SPC6)
to the growing medium. Bands 7 and 8 correspond to medium
conditioned by genetically modified H6A cells to produce and
secrete a new and yet not characterized isoform of PACE4 (band 7
H6A-PACE4) and SPC6 (band 8 H6A-SPC6).
[0124] Band 4 corresponds to the medium conditioned by H6A cells
growing with the medium conditioned by genetically modified
CHO-DG44 cells only with the expression vector of endoproteases
(H6A +CHO-DG44-EGFP).
[0125] One can observe on FIG. 8 that the 100 kDa proteic band
corresponding to non-processed light chain just in the medium
conditioned by H6A cells growing with the medium conditioned by
CHO-DG44-SPC6-EGFP cells (band 6) has disappeared, indicating that,
upon the addition of SPC6, in this case produced by another cell
line, to the growing medium of cells producing Factor VIII,
proteolytic processing of the light chain takes place. The fact the
processing has been partial in this assay is probably due to the
use of insufficient quantities of SPC6 to fully perform this
processing.
[0126] When this endoprotease (SPC6) is produced simultaneously
with Factor VIII, processing of the light chain is full (band
8).
[0127] It should be realized that the above disclosed embodiments
are merely illustrative and any change throughout them may occur
for an expert in the art. Consequently, the present invention
should not be considered as limited to the embodiments disclosed
herewithin.
[0128] The expert in the art will know how to promptly evaluate, by
means of the teachings as included in the text and in the examples
the advantages of the invention and propose variations and
equivalent embodiment alternatives, not however escaping from the
scope of the invention as defined by the attached claims.
* * * * *